Achieving high imaging speed is critical to enabling the incorporation of digital imaging in a clinical workflow. Digital slide scanning, or whole slide imaging (WSI), is increasingly used in clinical environments owing to the benefits it provides in terms of remote interpretation by experts, security and longevity for archiving, potentially higher review efficiency, and amenability to evolving tools for computer aided diagnosis. An important measure of functionality is the throughput of the digitalization instrument, most often quoted in terms of time required to scan a 15 mm×15 mm area and typically on the order of one to six minutes. Since slides are usually imaged serially on a given instrument, high throughput is desirable not only for ensuring pathologists have access to images as quickly as possible, but (perhaps most importantly) for reducing the cost-per-sample of the scanning instrument which may otherwise be prohibitively high.
Multiphoton microscopes (MPM) (e.g., as described in U.S. Pat. No. 5,034,613 to Denk et al.) have proven to be a valuable tool for biological research and offer a potential alternative for histologic analysis (and WSI) in clinical and research settings. In order to allow future clinical implementation of MPM in histologic analysis, existing speed limitations must be critically addressed.
Various aspects contribute to achievable image acquisition speeds in MPM. Systems typically comprise a laser source that produces ultrashort laser pulses of about 50 fs-2 ps, with a repetition rate of ˜80 MHz. The light from the laser is focused by a microscope objective to a point inside the sample. This point is scanned across the sample by a system of mirrors placed upstream of the objective that may be mounted on galvanometers. The generated fluorescence is typically collected back through the objective lens and directed to one or more detectors by a series of dichroic and emission filters, or by collection optics positioned on the opposite side of the sample from the objective lens and directed to one or more detectors by a series of dichroic and emission filters. The generation of fluorescence in the sample occurs by the simultaneous absorption of two or more photons from the laser. The use of short pulses leads to high peak intensities for more efficient multiphoton absorption without requiring excessive average laser powers.
Galvanometers used for point scanning are typically comprised of mirrors mounted on shafts that rotate in either a linear or sinusoidal fashion, deflecting the beam in a line pattern. The typical multiphoton microscope scans one line of the sample in ˜1 ms, with pixel dwell times on the order of ˜1 us. The pixel clock, which determines when signal coming from the detectors gets assigned to a new pixel, is synced to the position of the scanning optics in order to create pixels of uniform size. Because the pixel dwell time is long compared to the time between laser pulses (typically 12 ns), many pulses will strike the sample during a given typical pixel dwell. It is therefore unimportant to count the number of pulses arriving per pixel. However, when imaging at much higher scan rates than the typical system, pixel dwell times may shorten to the point that the variation in the number of pulses per pixel leads to unwanted variation in the amount of fluorescence collected from pixel to pixel.
For microscopes that incorporate at least one resonant galvanometer for high-speed scanning, the non-linear, sinusoidal scan pattern of the resonant galvanometer results in a very large variation in the number of pulses per pixel. This creates pronounced inhomogeneity in image intensity across the field-of-view and limits the maximum rate that can be achieved for a given minimum number of pulses per pixel. The latter occurs because the sinusoidal movement of the mirror results in pixel collection that is slower at the edges of the field of view than at the midpoint of rotation where the speed is at its maximum. The inhomogeneity in image intensity translates to image quality limitations that limit the applicability of resonant galvanometer based MPM systems to diagnostic interpretation of tissue histology.
Spinning polygons have also been used to increase the rate of line scanning in point-scanning systems and do not suffer from the variable speed issues of standard or resonant galvanometers. Shack et al. (1979) described theoretical speed optimization using continuous wave lasers and spinning polygons for point scan imaging of biological samples by coupling it with continuous motion of perpendicularly oriented stage movement. The description refers to the potential for fast imaging, but beyond being theoretical, the description predates the invention of the multiphoton microscope and thus does not address aspects specific to the coupling of spinning polygons to multiphoton excitation.
Some multiphoton microscopes have used high-repetition-rate laser pulse trains in order to increase the speed of imaging or to lower the peak power in order to reduce photobleaching and photodamage. Amir et al. 2007 used a beamsplitter and a delay line to double the effective repetition rate of 23 MHz laser pulses, while Cheng et al. 2011 used multiple beamsplitters, resulting in a 4-fold increase in the 80 MHz pulse rate of the source laser. However, both Amir and Cheng focused the outputs of the beamsplitters onto different spots within the sample, such that the effective pulse rate for a single spot was unchanged from that of the source laser. Chu et al. 2003 used an ultrafast laser with a 2 GHz repetition rate for second harmonic imaging, but did not use this laser for multiphoton fluorescence imaging. Ni et al. 2008 used a series of beamsplitters and delay lines to image with a single scanning spot with an effective pulse repetition rate of 640 MHz-10.24 GHz, much faster than the lifetime of the fluorescent protein they were imaging. While that approach worked for the specific samples used, its general applicability with other samples and dyes is questionable due to excited-state absorption resulting in increased dark state population and photobleaching. Thus, no solution presented to date has been able to achieve a MPM with maximized speed that ensures maximal quality for detailed histologic evaluation in a timely fashion.